【摘要】 目的:实现β-甘露聚糖酶基因在野生酵母菌中的整合诱导型表达。方法:克隆猪肠道野生酵母菌JSY4的25S rDNA片段;并与YIP5经EcoRⅠ与BamHⅠ双酶切连接,构建载体YIP5-rDNA。BamHⅠ与SalⅠ双酶切YIP5-rDNA,EcoRⅠ与SalⅠ双酶切pGEM-ManⅠ得到ManⅠ基因,BglⅡ与EcoRⅠ双酶切pPIC9K得到AOX1启动子,将三个片段连接,构建高拷贝整合诱导型表达载体YIP5-rDNA-AOX1-ManⅠ。提取DNA用SalⅠ单酶切线性化与pAX15按3∶1的比例共转化JSY4,在含300μg/mlG418的YEPD平板上筛选工程菌转化子,PCR方法鉴定。用2%甲醇诱导共转化工程菌以实现表达。结果:成功表达出β-甘露聚糖酶,其比活力为0.90IU/ml。而且传代10次后仍能检测到ManⅠ基因的表达产物β-甘露聚糖酶。结论:实现了β-甘露聚糖酶基因随共转化工程菌染色体稳定遗传及表达的目的。更多还原

【Abstract】 Objective: Expression β-Mannanase gene in wild type yeast.Methods: Cloned 25S rDNA fragment of JYS4 from wild type yeast in pig intestinal,then ligage it into vector YIP5 by enzyme EcoRⅠand BamHⅠ,and get a new vector YIP5-rDNA.Ligaged rDNA from YIP5-rDNA digested by BamHⅠand SalⅠ,ManⅠfrom vector pGEM-ManⅠ digested by EcoRⅠand SalⅠ,and promoter AOX1 from pPIC9K digested by BglⅡand EcoRⅠ at sane tine.A high clone integrated expression vector YIP5-rDNA-AOX1-ManⅠ was finished.Conversed JSY4 with DNA of YIP5-rDNA-AOX1-ManⅠ and pAX15 as the proportion of 3∶1.Transformants were screened in YEPD flat panel of 300μg/ml G418 by PCR.Tnducing by 2% methyl alcohol,the engineering yeast YIP5-rDNA-AOX1-ManⅠ successfully expression.Results: Successfully expressed the β-Mannanase that specific activity is 0.90 IU/ml.furthermore,It can examine ManⅠgene occuring after genesis ten times and achieve β-Mannanase expressing. Conclusion: Successful expression β-Mannanase gene in wide type yeast and it can stability hereditary.更多还原